Male Germ Cell Differentiation Research Articles

O-133 Retinoic acid has an essential role in meiotic progression beyond the zygotene stage in mouse testis

Abstract Study question Is all-trans retinoic acid (ATRA) instrumental to male germ cell differentiation in the mouse beyond the A1 spermatogonia cell stage? Summary answer While ATRA is useless to meiosis initiation, it is crucial to allow germ cells progressing beyond the mid-pachytene stage of meiosis. What is known already In mammals, ATRA is instrumental to spermatogenesis. It is synthesized by retinaldehyde dehydrogenases (RALDH), and then it binds to and activates nuclear receptors (RAR) either within the ATRA-synthesizing cells or in the neighboring cells. We established previously that ATRA synthesized by Sertoli cells initiates differentiation of A aligned into A1 spermatogonia during the 1st spermatogenic cycle. A controversy exists as to the requirement of ATRA in meiosis and spermiogenesis initiations. Some authors propose that ATRA is indispensable to both, while others claim that ATRA is useless to meiotic initiation but essential to spermiogenesis. Study design, size, duration In order to determine the role of ATRA in meiosis and spermiogenesis, we have generated mutant mice lacking all retinaldehyde dehydrogenase (RALDH) activities either in Sertoli cells or in the whole seminiferous epithelium (i.e., in both Sertoli and germ cells). We then compared in these two types of mutants the fate of spermatocytes generated during the first wave of spermatogenesis, following A1 spermatogonia differentiation at post-natal day 4. Participants/materials, setting, methods To generate these mutants, transgenic mice in which the Cre recombinase expression is driven by Stra8 or Amh promoters (active in undifferentiated spermatogonia and Sertoli cells, respectively) were crossed with mice bearing loxP-flanked alleles of Raldh1, Raldh2 and Raldh3. The animals bearing the Amh-Cre transgene lack ATRA only in Sertoli cells, while those bearing both the Amh-Cre and Stra8-Cre transgenes lack ATRA in the whole seminiferous epithelium. Main results and the role of chance In mice lacking RALDH in the whole seminiferous epithelium, we show that differentiated spermatogonia initiate meiosis properly, despite the evident lack of ATRA. However, the generated spermatocytes die by apoptosis at the mid-pachytene stage. They display signs of altered meiotic recombination (i.e., increased methylation of histone H3 on lysine 4, increased phosphorylation of histone H2AX variant, increased number of RAD51 foci), without abnormal chromosomal pairing, as assessed by SYCP1 and SYCP3 distributions. In addition, they lose TEX14 expression and inter-cytoplasmic connections, thus forming syncytia. When ATRA is missing in seminiferous epithelium, the Sertoli cells also are affected. They display an altered architecture, a disrupted distribution of connexin 43, and fail to maintain a functional blood-testis barrier. These defects are possibly related to a cell-autonomous, abnormal, increase of SRC kinase phosphorylation. We finally demonstrate that ATRA given at the preleptotene/leptotene stage is able to rescue all of these defects and to allow normal meiotic progression and spermiogenesis. Altogether, these results indicate that ATRA is required during the early step of meiotic prophase I, not to initiate meiosis by inducing STRA8 expression, but to allow proper chromosome recombination, survival of spermatocytes beyond the mid-pachytene stage and maintenance of Sertoli cell functioning. Limitations, reasons for caution These results were obtained using a mouse model and therefore the relevance for human spermatogenesis remains to be demonstrated. Wider implications of the findings Pharmacological studies support a role for ATRA in spermatogenesis in humans. Whether testicular impairment of ATRA synthesis or functioning might be a cause of azoospermia or oligozoospermia remains to be explored in clinical research. According to our study, ATRA emerges as a potential candidate for developing treatments in infertile patients. Trial registration number N.A.

TEX13B is essential for metabolic reprogramming during germ cell differentiation.

What is the functional significance of Tex13b in male germ cell development and differentiation? Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. N/A. We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

Small Non-Coding RNAs in Male Reproduction.

Male reproductive functions are strictly regulated in order to maintain sperm production and fertility. All processes are controlled by precise regulation of gene expression, which creates specific gene expression programs for different developmental stages and cell types, and forms the functional basis for the reproductive system. Small non-coding RNAs (sncRNAs) are involved in gene regulation by targeting mRNAs for translational repression and degradation through complementary base pairing to recognize their targets. This review article summarizes the current knowledge on the function of different classes of sncRNAs, in particular microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), during male germ cell differentiation, with the focus on sncRNAs expressed in the germline. Although transcriptionally inactive, mature spermatozoa contain a complex population of sncRNAs, and we also discuss the recently identified role of sperm sncRNAs in the intergenerational transmission of epigenetic information on father's environmental and lifestyle exposures to offspring. Finally, we summarize the current information on the utility of sncRNAs as potential biomarkers of infertility that may aid in the diagnosis and prediction of outcomes of medically assisted reproduction.

A medaka gonad-specific lncRNA may act as pri-miR-202 to regulate testicular endocrine homeostasis and spermatogenesis

A large number of long non-coding RNAs (lncRNAs) are expressed in animal gonads, but their functions are poorly understood. In this study, a gonad-specific lncRNA, termed lnc4, was identified and characterized in the model fish medaka (Oryzias latipes). The expression pattern and in vitro functional analyses indicated that lnc4 was likely to be a primary transcript of miR-202 (pri-miR-202). Results of single-molecule fluorescence in situ hybridization demonstrated that the precursor miR-202 (pre-miR-202) was highly expressed in the nuclei of testicular somatic cells, including Leydig and Sertoli cells, whereas only a small amount of lnc4 molecules could be detected co-expressed with pre-miR-202 in Sertoli cells due to its low expression level. Deletion of the lnc4 locus led to a significant reduction in testis size and a dramatic decrease in the number of male germ cells, as well as a reduction in sperm viability. Moreover, lnc4 knockout resulted in enhanced synthesis and secretion of testicular somatic cells and accelerated differentiation of immature male germ cells. Taken together, functional studies of lnc4 and its mature transcript miR-202 will contribute to the understanding of the important role of non-coding RNAs in animal or human reproductive disorders.

Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.

Single-Cell RNA Sequencing Reveals Atlas of Yak Testis Cells.

Spermatogenesis is a complex process that involves proliferation and differentiation of diploid male germ cells into haploid flagellated sperm and requires intricate interactions between testicular somatic cells and germ cells. The cellular heterogeneity of this process presents a challenge in analyzing the different cell types at various developmental stages. Single-cell RNA sequencing (scRNA-seq) provides a useful tool for exploring cellular heterogeneity. In this study, we performed a comprehensive and unbiased single-cell transcriptomic study of spermatogenesis in sexually mature 4-year-old yak using 10× Genomics scRNA-seq. Our scRNA-seq analysis identified six somatic cell types and various germ cells, including spermatogonial stem cells, spermatogonia, early-spermatocytes, late-spermatocytes, and spermatids in yak testis. Pseudo-timing analysis showed that Leydig and myoid cells originated from common progenitor cells in yaks. Moreover, functional enrichment analysis demonstrated that the top expressed genes in yak testicular somatic cells were significantly enriched in the cAMP signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and ECM receptor interactions. Throughout the spermatogenesis process, genes related to spermatogenesis, cell differentiation, DNA binding, and ATP binding were expressed. Using immunohistochemical techniques, we identified candidate marker genes for spermatogonial stem cells and Sertoli cells. Our research provides new insights into yak spermatogenesis and the development of various types of cells in the testis, and presents more reliable marker proteins for in vitro culture and identification of yak spermatogonial stem cells in the later stage.

Oxygen availability influences the incidence of testicular teratoma in Dnd1Ter/+ mice.

Testicular teratomas and teratocarcinomas are the most common testicular germ cell tumors in early childhood and young men, and they are frequently found unilaterally in the left testis. In 129/SvJ mice carrying a heterozygous copy of the potent modifier of tumor incidence Ter, a point mutation in the dead-end homolog one gene (Dnd1 Ter/+), ∼70% of the unilateral teratomas arise in the left testis. We previously showed that in mice, left/right differences in vascular architecture are associated with reduced hemoglobin saturation and increased levels of the hypoxia inducible factor-1 alpha (HIF-1α) in the left compared to the right testis. To test the hypothesis that systemic reduction of oxygen availability in Dnd1 Ter/+ mice would lead to an increased incidence of bilateral tumors, we placed pregnant females from 129/SvJ Dnd1 Ter/+ intercross matings in a hypobaric chamber for 12-h intervals. Our results show that in 129/SvJ Dnd1 Ter/+ male gonads, the incidence of bilateral teratoma increased from 3.3% to 64% when fetuses were exposed to acute low oxygen conditions for 12-h between E13.8 and E14.3. The increase in tumor incidence correlated with the maintenance of high expression of pluripotency genes Oct4, Sox2 and Nanog, elevated activity of the Nodal signaling pathway, and suppression of germ cell mitotic arrest. We propose that the combination of heterozygosity for the Ter mutation and hypoxia causes a delay in male germ cell differentiation that promotes teratoma initiation.

Revisiting the gonadotropic regulation of mammalian spermatogenesis: evolving lessons during the past decade.

Spermatogenesis is a multi-step process of male germ cell (Gc) division and differentiation which occurs in the seminiferous tubules of the testes under the regulation of gonadotropins - Follicle Stimulating Hormone (FSH) and Luteinising hormone (LH). It is a highly coordinated event regulated by the surrounding somatic testicular cells such as the Sertoli cells (Sc), Leydig cells (Lc), and Peritubular myoid cells (PTc). FSH targets Sc and supports the expansion and differentiation of pre-meiotic Gc, whereas, LH operates via Lc to produce Testosterone (T), the testicular androgen. T acts on all somatic cells e.g.- Lc, PTc and Sc, and promotes the blood-testis barrier (BTB) formation, completion of Gc meiosis, and spermiation. Studies with hypophysectomised or chemically ablated animal models and hypogonadal (hpg) mice supplemented with gonadotropins to genetically manipulated mouse models have revealed the selective and synergistic role(s) of hormones in regulating male fertility. We here have briefly summarized the present concept of hormonal control of spermatogenesis in rodents and primates. We also have highlighted some of the key critical questions yet to be answered in the field of male reproductive health which might have potential implications for infertility and contraceptive research in the future.

Endocannabinoid system and epigenetics in spermatogenesis and testicular cancer.

In mammals, male germ cell development starts during fetal life and is carried out in postnatal life with the formation of sperms. Spermatogenesis is the complex and highly orderly process during which a group of germ stem cells is set at birth, starts to differentiate at puberty. It proceeds through several stages: proliferation, differentiation, and morphogenesis and it is strictly regulated by a complex network of hormonal, autocrine and paracrine factors and it is associated with a unique epigenetic program. Altered epigenetic mechanisms or inability to respond to these factors can impair the correct process of germ development leading to reproductive disorders and/or testicular germ cell cancer. Among factors regulating spermatogenesis an emerging role is played by the endocannabinoid system (ECS). ECS is a complex system comprising endogenous cannabinoids (eCBs), their synthetic and degrading enzymes, and cannabinoid receptors. Mammalian male germ cells have a complete and active ECS which is modulated during spermatogenesis and that crucially regulates processes such as germ cell differentiation and sperm functions. Recently, cannabinoid receptor signaling has been reported to induce epigenetic modifications such as DNA methylation, histone modifications and miRNA expression. Epigenetic modifications may also affect the expression and function of ECS elements, highlighting the establishment of a complex mutual interaction. Here, we describe the developmental origin and differentiation of male germ cells and testicular germ cell tumors (TGCTs) focusing on the interplay between ECS and epigenetic mechanisms involved in these processes.

Parameters of spontaneous and benzene-induced cell death of spermatozoa under in vitro conditions in male employees of an oil production enterprise with impaired fertility

Introduction. Every year there is a steady increase in the frequency of male infertility both in Russia and in other countries of the world with the concomitant low efficiency of recognizing the causes of its idiopathic forms. The explanation for the increase in infertility are: harmful production factors, including chemical ones, progressive deterioration of the quality of the urban environment, which has a significant impact on the reproductive health of the urbanized population, recurring economic crises that negatively affect the human psyche, mass consumption of substandard products and medicines. These factors, primarily chemical production, negatively affect the reproductive system, including the differentiation of male germ cells, as a cause of fertility disorders. The aim of the study to consider in vitro spontaneous and benzene-induced features of sperm apoptosis in male oil industry workers with impaired fertility. Materials and methods. We have conducted an experimental study of biological material (ejaculate) in 50 men with reproductive disorders (26-49 years old), employees of an oil production enterprise. To conduct the experiment in vitro, the researchers used seminal fluid samples. A benzene solution at a concentration of 0.001 micrograms/ml was used as an in vitro induction factor (selected experimentally). We divided the analyzed samples into 2 equal parts to study the spontaneous and benzene-induced expression of apoptosis markers (CD25+, CD95+, bax, p53, Annexin V-FITC and Propidium Iodide, Caspasa-3), which the researchers determined by flow cytometry. Results. There were no significant deviations of intracellular markers of sperm apoptosis (bax, p53, Annexin V-FITC+7AAD+, Annexin V-FITC+7AAD-, Caspasa-3), however, experts observed differences in the expression of CD95+ and CD25+ membrane proteins by 20% and 10% compared to the spontaneous level, which is associated with the acceleration of the events of programmed sperm death (p<0.05). Conclusion. The results obtained in an in vitro experiment on modeling the apoptosis program by induction with hapten (benzene) showed that violations of sperm cell death are associated with overexpression of the key receptor for triggering apoptosis CD95, as well as a marker of early activation of CD25 cells, which reflects benzene modification of the cell death scenario along the path of its excessive stimulation.

SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.

Bud31-mediated alternative splicing is required for spermatogonial stem cell self-renewal and differentiation.

Alternative splicing (AS) is tightly regulated during cell differentiation and development. AS events are prevalent in the testis, but the splicing regulation in spermatogenesis remains unclear. Here we report that the spliceosome component Bud31 plays a crucial role during spermatogenesis in mice. Germ cell-specific knockout of Bud31 led to loss of spermatogonia and to male infertility. We further demonstrate that Bud31 is required for both spermatogonial stem cell pool maintenance and the initiation of spermatogenesis. SMART-seq revealed that deletion of Bud31 in germ cells causes widespread exon-skipping and intron retention. Particularly, we identified Cdk2 as one of the direct splicing targets of Bud31, knockout of Bud31 resulted in retention of the first intron of Cdk2, which led to a decrease in Cdk2 expression. Our findings suggest that Bud31-mediated AS within spermatogonial stem cells regulates the self-renewal and differentiation of male germ cells in mammals.

MiR-106b enhances human mesenchymal stem cell differentiation to spermatogonial stem cells under germ cell profile genes involved in TGF-b signaling pathways.

Mesenchymal stem cells can be differentiated into tissue-specific cells. MicroRNAs (miRNAs) regulate the translation of mRNAs involved in the growth and development of a variety of cells, including primordial germ cells (PGCs). This study evaluated male germ cell differentiation from human MSCs by miR-106b. The MSCs were obtained from human adipose tissue. The differentiation of MSCs into PGCs was accomplished by transfection of a lentiviral vector expressing miR-106b. MSCs were treated with bone morphogenic factor 4 as a control and also as a putative inducer of PGC differentiation. PGC was differentiated into spermatogonial-like cells by retinoic acid. Moreover, Dazl, Plzf, Stra8, Gfra, and Thy1 gene expressions were investigated using real-time PCR. Our results showed that Dazl, Plzf, and Stra8 genes that were treated with BMP4 and miR-106b did not show any significant difference, meaning that miR-106b, like BMP4, is able to differentiate PGC cells from MSCs. In spermatogonial-like cells, Thy1 was significantly unregulated in both the miR-106b and BMP4 groups. Our findings showed that miR-106b regulates the differentiation of MSCs into PGCs. miR-106b influences on the expression of Dazl, Plzf, and Stra8 genes in PGC and Gfra, Stra8, and Thy1 genes.

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

BackgroundGerm cell mitotic arrest is conserved in many vertebrates, including birds, although the time of entry or exit into quiescence phase differs. Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch. However, mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage.ResultsWe performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens. Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages (E12, E16, and hatch), we found that metabolic and signaling pathways were regulated, and that the epigenome was reprogrammed during mitotic arrest. In particular, we found that histone H3K9 and H3K14 acetylation (by HDAC2) and DNA demethylation (by DNMT3B and HELLS) led to a transcriptionally permissive chromatin state. Furthermore, we found that global DNA demethylation occurred gradually after the onset of mitotic arrest, indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome. DNA hypomethylation persisted after hatching, and methylation was slowly re-established 3 weeks later.ConclusionsWe found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching. This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described. Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

Coding and regulatory transcriptome comparisons between fertile and infertile spermatozoa identify RNA signatures of male infertility

The aim of the present study was to identify RNA-based signatures of male infertility by sperm transcriptome analysis. In this study, deep sequencing analyses of coding (mRNA) and regulatory (miRNA) transcriptomes were performed by pooling 15 oligo/oligoasthenozoospermic infertile sperm and 9 normozoospermic fertile sperm samples. Furthermore, interesting candidates were selected for validation by real-time PCR. The comparison of miRNAs between cases and controls identified 94 differentially expressed miRNAs, of which at least 38 have known functions in spermatogenesis. In transcriptome (mRNA) data, a total of 60,505 transcripts were obtained. The comparison of coding RNAs between cases and controls revealed 11,688 differentially expressed genes. miRNA-mRNA paired analysis revealed that 94 differentially expressed miRNAs could potentially target 13,573 genes, of which 6419 transcripts were actually differentially expressed in our data. Out of these, 3303 transcripts showed inverse correlation with their corresponding regulatory miRNAs. Moreover, we found that most of the genes of miRNA-mRNA pairs were involved in male germ cell differentiation, apoptosis, meiosis, spermiogenesis and male infertility. In conclusion, we found that a number of sperm transcripts (miRNAs and mRNAs) have a very high potential of serving as infertility/sperm quality markers.

Overexpression of bmp4, dazl, nanos3 and sycp2 in Hu Sheep Leydig Cells Using CRISPR/dcas9 System Promoted Male Germ Cell Related Gene Expression.

Simple SummaryMale germ cell development plays a crucial role in male reproduction, and gene expression also presents an essential regulatory role in its development. Many studies have been devoted to the induction and differentiation of pluripotent stem cells into germ cells in vitro. However, the culture system for pluripotent stem cells from domestic animals is not stable, especially in sheep. Our study attempted to transdifferentiate sheep somatic cells into germ cells in vitro by the overexpression of key germ cell related genes, with the aim of perfecting the construction of germ cell research models in vitro. Therefore, we explored the expression pattern of four crucial genes, bmp4, dazl, nanos3 and sycp2, in Hu sheep testicular development, and investigated the potential efficiency of overexpression of the four candidate genes using the CRISPR/dcas9 system in Leydig cells. We revealed that the overexpression of bmp4, dazl, nanos3 and sycp2 can promote the expression of male germ cell related genes. To the best of our knowledge, this is the first study to construct an overexpression induction system using CRISPR/dcas9 technology, and to induce sheep somatic cells into germ cells in vitro.Male germ cells directly affect the reproduction of males; however, their accurate isolation and culture in vitro is extremely challenging, hindering the study of germ cell development and function. CRISPR/dcas9, as an efficient gene reprogramming system, has been verified to promote the transdifferentiation of pluripotent stem cells into male germ cells by editing target genes. In our research, we explored the expression pattern of the germ cell related genes bmp4, dazl, nanos3 and sycp2 in Hu sheep testicular development and constructed the overexpression model using the CRISPR/dcas9 system. The results indicated that four genes showed more expression in testis tissue than in other tissues, and that bmp4, dazl and sycp2 present higher expression levels in nine-month-old sheep testes than in three-month-olds, while nanos3 expressed the opposite trend (p < 0.05). In addition, the expression of four potential genes in spermatogenic cells was slightly different, but they were all expressed in sheep Leydig cells. To verify the potential roles of the four genes in the process of inducing differentiation of male germ cells, we performed cell transfection in vitro. We found that the expression of the germ cell related genes Prdm1, Prdm14, Mvh and Sox17 were significantly increased after the overexpression of the four genes in Leydig cells, and the co-transfection effect was the most significant (p < 0.05). Our results illustrate the crucial functions of bmp4, dazl, nanos3 and sycp2 in Hu sheep testis development and verified the effectiveness of the overexpression model that was constructed using the CRISPR/dcas9 system, which provided a basis for further male germ cell differentiation in vitro.

Post-transcriptional regulation in spermatogenesis: all RNA pathways lead to healthy sperm.

Multiple RNA pathways are required to produce functional sperm. Here, we review RNA post-transcriptional regulation during spermatogenesis with particular emphasis on the role of 3' end modifications. From early studies in the 1970s, it became clear that spermiogenesis transcripts could be stored for days only to be translated at advanced stages of spermatid differentiation. The transition between the translationally repressed and active states was observed to correlate with the shortening of the transcripts' poly(A) tail, establishing a link between RNA 3' end metabolism and male germ cell differentiation. Since then, numerous RNA metabolic pathways have been implicated not only in the progression through spermatogenesis, but also in the maintenance of genomic integrity. Recent studies have characterized the elusive 3' biogenesis of Piwi-interacting RNAs (piRNAs), identified a critical role for messenger RNA (mRNA) 3' uridylation in meiotic progression, established the mechanisms that destabilize transcripts with long 3' untranslated regions (3'UTRs) in post-mitotic cells, and defined the physiological relevance of RNA exonucleases and deadenylases in male germ cells. In this review, we discuss RNA processing in the male germline in the light of the most recent findings. A brief recollection of different RNA-processing events will aid future studies exploring post-transcriptional regulation in spermatogenesis.

The Effect of Bisphenol A on Notch Signaling Pathway in Development of Rat Testis

Bisphenol A (BPA) is the object of great concerns because of its widespread use, due to several millions of tons its production throughout the world each year. BPA is an endocrine disrupting chemical and is classified as a probable human carcinogen. Fetal and perinatal exposure to environmentally relevant doses of BPA induces multigenerational impairments of male fertility. Notch signaling functions to regulate cell-fate by modulating differentiation, proliferation, and survival of cells. Notch signaling system plays a significant role in male germ cell differentiation and survival. In this study, it was aimed to explore the alterations/changes in protein expression of Notch I receptor following BPA treatments during embriyonal (E) days 18 to 21, postnatal (P) days 0 to 3, and P4 to P7 time intervals in the rat testes in vivo. The results of study revealed that the used doses of BPA had’nt effect on Notch 1 protein expressions in rat testes during selected time preiods.

Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis

Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in the seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.

The therapeutic potential ofadipose tissue-derived mesenchymal stromal cellsin thetreatmentof busulfan-induced azoospermic mice.

The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.